Bottom Line:
The rtL269I substitution alone did not confer resistance to LMV, ETV, adefovir (ADV), or tenofovir (TDF).The clinical relevance of the rtL269I substitution was validated by its emergence in association with YMDD mutation in chronic hepatitis B (CHB) patients with sub-optimal response or treatment failure to LMV or CLV.Our study suggests that substitution at rt269 in HBV polymerase is associated with multi-drug resistance, which may serve as a novel compensatory mutation for replication-defective multi-drug resistant HBV.

Affiliation: Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea.

ABSTRACTThe emergence of compensatory mutations in the polymerase gene of drug resistant hepatitis B virus (HBV) is associated with treatment failure. We previously identified a multi-drug resistant HBV mutant, which displayed resistance towards lamivudine (LMV), clevudine (CLV), and entecavir (ETV), along with a strong replication capacity. The aim of this study was to identify the previously unknown compensatory mutations, and to determine the clinical relevance of this mutation during antiviral therapy. In vitro mutagenesis, drug susceptibility assay, and molecular modeling studies were performed. The rtL269I substitution conferred 2- to 7-fold higher replication capacity in the wild-type (WT) or YMDD mutation backbone, regardless of drug treatment. The rtL269I substitution alone did not confer resistance to LMV, ETV, adefovir (ADV), or tenofovir (TDF). However, upon combination with YMDD mutation, the replication capacity under LMV or ETV treatment was enhanced by several folds. Molecular modeling studies suggested that the rtL269I substitution affects template binding, which may eventually lead to the enhanced activity of rtI269-HBV polymerase in both WT virus and YMDD mutant. The clinical relevance of the rtL269I substitution was validated by its emergence in association with YMDD mutation in chronic hepatitis B (CHB) patients with sub-optimal response or treatment failure to LMV or CLV. Our study suggests that substitution at rt269 in HBV polymerase is associated with multi-drug resistance, which may serve as a novel compensatory mutation for replication-defective multi-drug resistant HBV.

pone.0136728.g001: Characterization of multi-drug resistant HBV mutant 50â2 isolated from a chronic HBV patient.(A) Schematic representation of the mutations in RT and overlapping surface gene of clone 50â2. The amino acid changes in polymerase and corresponding surface gene are indicated by arrows. (B) Comparison of intracellular and extracellular secreted HBV DNA levels between WT virus and clone 50â2. The HBV 1.2mer construct plasmids (2 Îžg) were transfected into Huh7 hepatoma cells and harvested at 72h post-transfection. The linearized 3.2kb HBV genome was loaded in lane 1 as a marker. (C) & (D) In vitro susceptibility WT HBV (C) and clone 50â2 (D) to lamivudine (LMV), clevudine (CLV), entecavir (ETV), adefovir (ADV), and tenofovir (TDF). Cells were treated for 3 days with each drug, and the replication level was compared with that of the WT (without drug treatment). The relative HBV replication level was quantified using Phosphorimager. The standard deviation of three independent experiments was measured (***, P < 0.001).

Mentions:
We have previously reported a multi-drug resistant HBV mutant (clone 50â2), which harbored the quintuple rtM129L+V173L+M204I+L269I+H337N mutations in the RT domain [15]. Due to the overlap of the polymerase gene with the surface gene, these mutations are accompanied by two mutations in the surface gene; sE164D and sW196L (Fig 1A). The average half-maximal inhibitory concentration (IC50) values of clone 50â2 were determined to be >100, >100, 0.7, 3.5, and 5.9 ÎžM for LMV, CLV, ETV, ADV, and TDF, respectively [15]. In order to characterize this mutant in detail, its replication ability and drug susceptibility were further analyzed (Fig 1).

pone.0136728.g001: Characterization of multi-drug resistant HBV mutant 50â2 isolated from a chronic HBV patient.(A) Schematic representation of the mutations in RT and overlapping surface gene of clone 50â2. The amino acid changes in polymerase and corresponding surface gene are indicated by arrows. (B) Comparison of intracellular and extracellular secreted HBV DNA levels between WT virus and clone 50â2. The HBV 1.2mer construct plasmids (2 Îžg) were transfected into Huh7 hepatoma cells and harvested at 72h post-transfection. The linearized 3.2kb HBV genome was loaded in lane 1 as a marker. (C) & (D) In vitro susceptibility WT HBV (C) and clone 50â2 (D) to lamivudine (LMV), clevudine (CLV), entecavir (ETV), adefovir (ADV), and tenofovir (TDF). Cells were treated for 3 days with each drug, and the replication level was compared with that of the WT (without drug treatment). The relative HBV replication level was quantified using Phosphorimager. The standard deviation of three independent experiments was measured (***, P < 0.001).

Mentions:
We have previously reported a multi-drug resistant HBV mutant (clone 50â2), which harbored the quintuple rtM129L+V173L+M204I+L269I+H337N mutations in the RT domain [15]. Due to the overlap of the polymerase gene with the surface gene, these mutations are accompanied by two mutations in the surface gene; sE164D and sW196L (Fig 1A). The average half-maximal inhibitory concentration (IC50) values of clone 50â2 were determined to be >100, >100, 0.7, 3.5, and 5.9 ÎžM for LMV, CLV, ETV, ADV, and TDF, respectively [15]. In order to characterize this mutant in detail, its replication ability and drug susceptibility were further analyzed (Fig 1).

Bottom Line:
The rtL269I substitution alone did not confer resistance to LMV, ETV, adefovir (ADV), or tenofovir (TDF).The clinical relevance of the rtL269I substitution was validated by its emergence in association with YMDD mutation in chronic hepatitis B (CHB) patients with sub-optimal response or treatment failure to LMV or CLV.Our study suggests that substitution at rt269 in HBV polymerase is associated with multi-drug resistance, which may serve as a novel compensatory mutation for replication-defective multi-drug resistant HBV.

Affiliation:
Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea.

ABSTRACTThe emergence of compensatory mutations in the polymerase gene of drug resistant hepatitis B virus (HBV) is associated with treatment failure. We previously identified a multi-drug resistant HBV mutant, which displayed resistance towards lamivudine (LMV), clevudine (CLV), and entecavir (ETV), along with a strong replication capacity. The aim of this study was to identify the previously unknown compensatory mutations, and to determine the clinical relevance of this mutation during antiviral therapy. In vitro mutagenesis, drug susceptibility assay, and molecular modeling studies were performed. The rtL269I substitution conferred 2- to 7-fold higher replication capacity in the wild-type (WT) or YMDD mutation backbone, regardless of drug treatment. The rtL269I substitution alone did not confer resistance to LMV, ETV, adefovir (ADV), or tenofovir (TDF). However, upon combination with YMDD mutation, the replication capacity under LMV or ETV treatment was enhanced by several folds. Molecular modeling studies suggested that the rtL269I substitution affects template binding, which may eventually lead to the enhanced activity of rtI269-HBV polymerase in both WT virus and YMDD mutant. The clinical relevance of the rtL269I substitution was validated by its emergence in association with YMDD mutation in chronic hepatitis B (CHB) patients with sub-optimal response or treatment failure to LMV or CLV. Our study suggests that substitution at rt269 in HBV polymerase is associated with multi-drug resistance, which may serve as a novel compensatory mutation for replication-defective multi-drug resistant HBV.